EP0008432A1 - Immunological determination process - Google Patents

Abstract

What is disclosed is a method for the immunological determination of an immuno-complex, comprising an antigen and an antibody, in a liquid containing said immuno-complex, which method comprises (a) incubating said liquid containing said immumo-complex with a first reagent bound to a carrier, said first reagent being specific for an antigenic determinant of the antigen in said immuno-complex and being present in an amount sufficient to fix said immuno-complex; (b) separating the carrier from said liquid containing said immuno-complex and incubating it in a solution of a second reagent, said second reagent being specific for an antigenic determinant of the antibody in said immuno-complex and being present in an amount sufficient to fix said immuno-complex; and (c) separating the carrier from said solution of the second reagent and determining the amount of bound or unbound second reagent.

Description

The invention relates to an immunological method for determining an immune complex formed from antiqen and antibodies in liquids.

_I mmunkomplexe are antigen-antibody compounds which are formed not only in vitro but also in vivo in many diseases and occur in body fluids. The antigen of an immune complex can be a representative of a variety of substances. These are often formed by microorganisms, viruses, bacteria, parasites, protozoa and occur in the bloodstream of higher life forms. However, they can also be products of animal tissue or of malignant cells which give rise to antibody formation as antigens. The antibodies induced by an antigen are specific to the antigen. Antibody formation in the living organism leads to the formation of antigen-antibody complexes, which are referred to as immune complexes. Immune complexes are able to bind complement factors in vivo. Immune complexes are usually quickly eliminated from the organism through the reticuloendothelial system. Under certain circumstances, immune complexes persist in the body and cause chronic immune complex diseases. ^p Resisting immune complexes are normally composed in such a way that not all immunological binding sites of the antigen are saturated with antibodies. The detection of immune complexes in body fluids provides valuable information for the diagnosis of diseases that can be summarized as immune complex diseases. In such diseases, however, not only the detection of immune complexes, but also the differentiation of the antigens contained in them is of great interest. Accordingly, there is a need for immune complex differentiation with regard to the antigen. So far, a two-step procedure was possible for this, namely first the detection of the immune complex and then the identification of the antigen.

It has now been found that the detection of immune complexes and the identification of the antigen can be achieved in a single procedure. There are basically two ways to do this.

• 1. Specific reactants A (usually antibodies) directed against the antigen of an immune complex are bound to a support. This coated carrier material is brought into contact with the liquid to be tested. Thereafter, the carrier treated in this way is brought into contact with a solution of an optionally labeled reactant B (antibody, complement), which is directed against the antibody present in the immune complex, and finally the bound or the portion of the reactant B dissolved in the liquid, preferably by Measurement of the marking agent, determined.
• 2. Instead of the antibody directed against the antigen in the immune complex, reactants A specific for the Fc part of the antibodies bound to the immune complex can be bound to a support. Such reactants are, for example, anti-Fc antibodies, anti-immunoglobulin aggregate antibodies, rheumatoid factors and complement factors. Subsequently, the carrier-bound immune complex with a solution of an optionally labeled reactane ten (antibody), directed against the antigen in the immune complex, brought into contact and the measurement was carried out as under 1.

In the case of a labeled reactant B, the amount of the particular labeling agent in each case indicates the amount of the immune complexes bound in each case. The labeling agent can be, for example, a radioactive atom, an enzyme, a coenzyme or a fluorescent group. Both methods are common that the antigen specificity is guaranteed by the use of antigen-specific antibodies. For example, anti-HBsAg antibodies are required for the detection of HBsAg-containing immune complexes, and anti-tetanus toxoid antibodies for the detection of immune complexes containing tetanus toxoid.

As an alternative to using labeled reactants, the carrier-bound immune complexes themselves can be detected by indicator reactions. This can e.g. by measuring complement binding or by agglutination of the carrier.

• a) ein für eine antigene Determinante des Antigens oder des Antikörpers spezifischer, an einen Träger gebundener Reaktant A in einerfür die Bindung des Immunkomplexes ausreichenden Menge mit der immunkomplexhaitigen Flüssigkeit inkubiert wird;
• b) der so behandelte Träger nach Abtrennung der Flüssi^qkeit mit einer für die Bindung an den Immunkomplex ausreichenden Menge einer Lösung eines gegebenenfalls markierten Reaktanten B inkubiert wird, der spezifisch für eine antigene Determinante des Antikörpers oder des Antigens ist;
• c) und danach durch Messung des gebundenen oder ungebundenen Reaktanten B der Immunkomplex in der Flüssigkeit bestimmt wird.

The invention accordingly relates to an immunological determination method for an immune complex in a liquid, characterized in that

• a) a reactant A, which is specific for an antigenic determinant of the antigen or of the antibody and is bound to a carrier, is incubated with the liquid containing the immune complex in an amount sufficient for binding the immune complex;
• b) the support thus treated after separation of the Flüssi ^q ness with a sufficient binding to the immunocomplex amount of a solution of an optionally labeled reactant B is incubated, which is specific for an antigenic determinant of the antibody or the antigen;
• c) and then by measuring the bound or unbound reactant B the immune complex in the liquid is determined.

A reactant A reacting with the antigen or a reactant A reacting with the antibody can thus be bound to the support. Accordingly, reactant _{B can be} specific for the antibody or for the antigen of the immune complex.

The invention further relates to a diagnostic agent for antigen-specific immune complex determination, characterized in that it contains carrier-bound reactants specific for the antigen or the antibody of the immune complex.

General procedure description

A reactant A specific for the antigen present in the immune complex or for the activated Fc part of the immune complex-bound antibody is bound to a solid support. Reactants specific for the antigen are antibodies or their antigen-binding fragments. Suitable reactants which are specific for the Fc part of immunocomplex-bound antibodies are mentioned above. Since immune complexes usually bind complement factors already in vivo, anti-complement antibodies, e.g. Fc-specific reactants can be used.

Suitable supports are shaped bodies, for example amorphous particles, spheres, platelets, foils or test tubes made of inorganic (for example glass) or organic material (for example homo- or copolymers of vinyl compound such as olefins, vinyl acetate, vinyl chloride, vinylidene chloride, tetrafluoroethylene, styrene, acrylic acid or methacrylic acid, also polymers of formaldehyde and cyclic acetals and of polycondensates, such as polyesters, polycarbonates or polyamides or crosslinked carbohydrates and crosslinked proteins); biological particulate carriers such as cells (e.g. erythrocytes).

The bond can be adhesive or covalent. The carrier is brought into contact with the reactant for adhesive coating. For example, the reactant is dissolved in an aqueous solution, preferably in a buffer (for example phosphate-buffered isotonic saline (PBS), pH 7.2), in a concentration of 1 μg to 1 g / l, preferably 10 μg / ml, for 1 min allowed to stand with the carrier for up to 5 days, preferably 24 hours. Excess reagent is removed by washing, advantageously with a buffer which suitably contains a detergent (e.g. PBS, pH 7.2 - polyoxyethylene sorbitan monolaurate (Tween 20) 0.1%).

Covalent bonds of the respective reactants are carried out with the different supports according to methods known from the literature (e.g. Orth et al., Angw. Chemie, 1972, 84, pp. 319 ff; Silman et al., Annu. Rev. Biochem., 1966, 35, Pp. 873 ff; Becht et al., J. Immunology, 1968, 101, pp. 18 ff; Weetall, Marcel Dekker, Inc., New York, p. 32, 1975).

The immune complexes are now determined as follows:

The carrier coated with the respective reactant A is incubated for 1 minute to 48 hours, preferably 10 hours, with the liquid containing immune complexes, preferably a serum sample, and is appropriately washed with buffer to remove excess serum components.

The carrier thus treated is then incubated with a solution of a preferably labeled reactant B, which is specific for the antibody bound to the immune complex or correspondingly for the antigen in the immune complex, for 1 minute to 48 hours, preferably for 1 hour. Reactants B that are specific for the antibody can be microbial protein A, complement, antibody or antibody fragments that are directed against antigenic determinants of native or denatured antibodies.

If the immune complex has bound complement factors in vivo, antibodies or antibody fragments which are directed against complement factors can also be used as antibody-specific reactants.

Reactants that are specific for the antigen are antigen-specific antibodies or antibody fragments. Specific antibody preparations are isolated either from the sera of specifically immunized animals or from human sera (e.g. anti-deoxyribonucleic acid antibodies from patient sera with lypus erythematosus, anti-hepatitis B antibodies from sera from patients with expired hepatitis, rheumatoid factors from patient sera with rheumatoid arthritis ) ( _L .: Humphrey and White: Immunologie, Thieme-Verlag Stuttgart, 1972; Gell, Coombs, Lachmann: Clinical Aspects of Immunology, Blackwell, Oxford, London, Edinburgh, Melburne, 1975). The optimal dilutions of the reactants to be used in the test must not enter into any unspecific connection with the support, ie a compound which can be detected without the presence of a carrier-bound immune complex. These optimal dilutions are to be determined for each reactant batch specifically by preliminary tests. Common dilutions are 1:10 to 1: 400. As a control, experiments are carried out which, as described above, are carried out with sera without immune complexes.

As already mentioned, preferred labeling agents for the reactants are, for example, radioactive and fluorescent substances or enzymes.

The measurement of the marking agent on the carrier can be carried out using the methods familiar to the person skilled in the art.

The invention is illustrated by the following examples:

Example 1:

Use of carrier-bound reactants A, specific

for the antigen tetanus toxoid.

Immune complexes with tetanus toxoid as an antigen are produced by mixing tetanus antibodies from humans with tetanus toxoid.

Preparation of a support coated with antibodies:

100 µl of a 1.1 x 10 ^-3 % solution (protein weight / volume of phosphate-buffered saline) of an anti-tetanus immunoglobulin from rabbit are filled into 400 µl polystyrene tubes and stored at 4 ° C. After 24 hours, the solution is suctioned off. The tubes are washed three times with 400 ul washing solution (phosphate buffered saline, pH 7.2, containing 0.1% (w / v) Tween 20).

1. Incubation:

The tubes coated with the anti-tetanus toxoid are filled with 0.1 ml of the immune complex-containing solution prepared as above, diluted 1: 4 with phosphate-buffered saline containing 5% bovine serum albumin, and left to stand at 21 ° C. for 10 hours. The solution is then suctioned off and washed as in the coating of the support.

2. Incubation:

Then 0.1 ml of one containing peroxidase according to the method of Nakane and Kawaoe, J.Histochem. Cytochem., 1972, 22, 1084) labeled rabbit anti-human gamma globulin at a dilution of 1:50 with phosphate buffered saline containing 5% bovine serum albumin, and it is then again after 1 hour (21 ° C) as in the coating washed.

3. Measurement of the marking:

0.1 ml of a freshly prepared 0.1% o-phenylenediamine HCl solution in citrate-phosphate buffer (5 ml of 0.1 M citric acid + 5 ml of 0.2 M Na ₂ HPO ₄ ), mixed with 0, 1 ml of a 1% (v / v) H ₂ O ₂ solution was pipetted into the tubes. After a reaction time of 30 to 60 minutes in the dark, the reaction is stopped with 0.1 ml of a 2 NH ₂ SO ₂ solution and the absorbance at 490 nm is measured using a Beckman photometer.

Immune complexes can be detected depending on their composition (antigen / antibody ratio), but particularly good in a slight excess of antigen.

Example 2:

Use of carrier-bound reactants A specific for the HBsAg antigen.

Patient sera with HBsAg-containing immune complexes and 60 normal sera were tested.

Production of the carrier coated with antibodies by adsorption:

100 µl of a 1.1 x 10 ^-3 % solution (weight protein / volume, phosphate-buffered saline) of an anti-hepatitis immunoglobulin from rabbit are filled into 400 µl polystyrene tubes. After 24 hours (4 ° C) the solution is suctioned off, washed three times with 400 ul phosphate-buffered saline, pH 7.2, containing 0.1% (w / v) Tween 20.

1. Incubation:

The tubes coated with the rabbit anti-hepatitis hyperimmunoglobulin are filled with 0.1 ml of the solution containing immune complexes, diluted 1: 4 with phosphate-buffered saline containing 5% bovine serum albumin, and left to stand at 21 ° C. for 10 hours.

The solution is then suctioned off and washed as in the coating of the support.

2. Incubation:

Then 0.1 ml of a rabbit anti-human gamma globulin labeled with peroxidase according to the method of Nakane and Kawaoe (J. Histochem. Cytochem., 1974, 22, 1084) is added in a dilution of 1:50 filled with phosphate-buffered saline containing 5% bovine serum albumin, and it is then washed again after 1 hour (21 ° C.) as in the coating of the support.

3. Measure the marking as in Example 1.

The results are shown in the table below

Antigen-specific immune complexes were clearly detected in sera from patients with hepatitis.

Example 3:

Use of polymethyl methacrylate film with active azide groups.

Coating the film:

Pieces of the same size with active azide groups from polymethylene methacrylate, produced by the method of M. Lynn, Immobilized Enzymes, Antigens, Antibodies and Peptides, ED Howard H. Weetall, Marcel Dekker, Inc., New York, 1975, p. 32) are incubated with a 0.1% solution of horse anti-tetanus toxoid immunoglobulin in PBS, pH 7.2 and washed three times with PBS, pH 7.2 to remove excess antibodies.

1. Incubation:

The film coated with horse antitoxoid is diluted 1: 4 with PBS albumin 5% with the immune complexes (immune complexes with tetanus toxoid as antigen as in Example 1) and incubated for 10 hours. The film is then washed three times with washing solution as in Example 1.

2. Incubation:

Subsequently, the film with peroxidase-labeled rabbit anti-human gamma globulin, 1:50 in PBS-BSA (see Example 1). diluted, incubated for 1 hour (21 ^° C.), washed three times with washing solution (see above) and the marking on the film was measured as in Example 1.

Results:

In the sample containing the immune complex, produced as in Example 1, antigen-specific immune complexes could be clearly detected compared to serum and tetanus toxoid without immune complexes.

Example 4:

Use of polymethyl methacrylate with active _A zidgruppen for the covalent binding of F (ab) ₂ fragments:

Coating the film: Polymethyl methacrylate film pieces of the same size (see Example 3) are prepared with a 0.1% solution of an anti-tetanus toxoid F (ab) ₂ horse preparation in PBS, pH 7.2 (produced by the Visonoff method incubated, A. et al. _A rch. Biochem. Biophys. 89, 230 (1960)) for 24 hours at 21 ⁰ C and to remove excess reactants 3 times with wash solution (s. Example 1) washed.

1. Incubation:

The coated film is incubated for 10 hours with the test sample containing immune complexes, which was diluted 1: 4 in PBS, pH 7.2 and BSA 5%, and then washed 3 times with washing solution.

2. Incubation:

The film is subsequently incubated in peroxidase-labeled (see Example 1) rabbit anti-human IgG, 1:50 diluted with PBS, pH 7.2 and BSA 5%, for 2 hours and then 3 times in washing solution (see Example 1) washed.

The marking on the film is measured by incubating the film pieces prepared in this way for 30-60 minutes in 0.5 ml of substrate solution (O-phenylenediamine · HCl solution, see Example 1) and then the absorbance of the solution at 490 nm measures.

Results:

Compared to the controls without immune complexes, the sample with immune complexes shows significantly higher extinction values.

Example 5:

Use of carrier-bound C1 as a reactant, specific for the antibody

Preparation of the coated support: 100 μl of human C1 _q (purified from human human serum according to Haupt, H. and Heimburger, N., Zeitschrift für physiol. Chem., 1972, 535, 1125) are placed in 400 μl polystyrene tubes. 0.1 %, dissolved in PBS, filled and left to stand at 4 ° C. for 24 hours. The polystyrene tubes are then washed three times with PBS containing 0.1% Tween 20.

1. Incubation:

The tubes treated in this way are filled with a test sample containing the HBsAg immune complexes (hepatitis _B- associated antigen), diluted 1: 4 with PBS and incubated for 10 hours at 4 ° C.

2. Incubation:

The tubes are treated with 0.1 ml of a rabbit anti-HBsAg immunoglobulin labeled with peroxidase by the method of Nakane and Kawaoe (see Example 1), 1: 150 in PBS, containing 5% bovine albumin, for 1 hour at 4 ° C incubated. The marking is measured as below

Example 1. Results:

The antigen-specific immune complex test shows significantly higher values (Factcr 2.7) compared to the control values in patient No. 2.

Example 6:

_V erwendunq of carrier-bound anti-Cl as a reactant, "specific" for the antibody

In 400 ul of a polystyrene tubes _at 0.1 ml t _{i q} -C1 immunoglobulin of rabbits, 0.1% ig filled in PBS and 24 hours at room temperature, allowed to stand. The tubes are then washed 3 times with washing solution (see above). The 1st and 2nd incubation and the measurement of the label are carried out as described in Example 5.

Results:

The results show clearly increased values in patient No. 2 (factor 33) compared to the control values.

Example 7:

Use of carrier-bound anti-human Fc antibody as a reactant

0.1 ml of an anti-Hüman Fc immunoglobulin from sheep, 0.1% in PBS, is poured into 400 μl polystyrene tubes and left to stand for 24 hours at room temperature. Then it is washed three times with washing solution (see above). The 1st and 2nd incubation and the measurement of the label are carried out as in Example 5.

Results:

The results show significantly increased values in patient serum No. 2 with immune complexes compared to control sera 1, 3.

Example 8:

According to the Becht method (J. Immunol. 101, p. 18, 1968), erythrocytes stabilized and tannelled with sulfosalicylic acid, 2% in PBS, are shaken with antitetanus toxoid immunoglobulin 0.01% from 24 hours at 4 ° C. Horse, incubated, then washed 3 times in PBS containing 0.1% Tween 20 and adjusted to a 1% cell suspension.

1. Incubation:

50 μl of the erythrocyte suspension are incubated with 50 μl test samples containing tetanus toxoid-human-antitoxoid-IC, diluted 1: 4 in PBS, pH 7.2, for 2 hours with shaking and then washed three times with washing solution.

2. Incubation:

The cell sediment is diluted 1:16 with 100 µl of freshly obtained guinea pig serum, incubated with shaking for 30 minutes at room temperature and then left to stand (10 min / 21 ° C) until the erythrocytes have settled on the tube bottom.

3. Measurement of complement consumption:

Fresh sheep erythrocytes are washed three times with PBS. _{1 ml Zellsed} i _men t is admixed with 0.5 ml of a 1% CrCl ₃ * suspended in _PBS and mixed with 0.5 ml of human immunoglobulin 1 _0%, washed three times with PBS after 4 minutes and adjusted to a 1% cell suspension. Equal volumes of 1% cell suspension and a rabbit _to tihuman IgG serum, 1% in PBS, are added together and incubated for 45 minutes at room temperature with shaking. *-Solution

50 μl of the cell suspension obtained in this way are left to stand for 30 minutes at 37 ° C. with 50 μl of the supernatant obtained after the second incubation. The absorbance of the cell-free supernatant is measured at 546 nm with a Beckman photometer. Low extraction values indicate a high consumption of complement in the second incubation step and thus a high level of immune complexes in the samples; high values mean that the test samples contain little immune complexes.

Results:

The results clearly show the antigen-specific detection of immune complexes, especially in immune complexes with a slight excess of antigen.

Claims (12)

1. Immunological determination method for an immune complex in a liquid, characterized in that a) a reactant A, which is specific to an antigenic determinant of the antigen or the antibody and is bound to a carrier, is incubated with the liquid containing the immune complex in an amount sufficient for binding the immune complex; b) after the liquid has been separated off, the carrier treated in this way is incubated with an amount of a solution of an optionally labeled reactant B which is specific for an antigenic determinant of the antibody or of the antigen and is sufficient for binding to the immune complex; c) and then by measuring the bound or unbound reactant B the immune complex in the liquid is determined. 2. The method according to claim 1, characterized in that the carrier is a biological particle or an organic or inorganic molding. 3. The method according to claim 1, characterized in that the binding of the reactant takes place covalently or adsorptively. 4. The method according to claim 1-3, characterized in that the reactant A reacting with the antigen is bound to the carrier and the reactant B reacting with the antibody carries a labeling agent. 5. The method according to claim 1-3, characterized in that the reactant A reacting with the antibody is bound to the carrier and the reactant B reacting with the antigen carries a labeling agent. 6. The method according to claim 1-5, characterized in that the reactant reacting with the antigen or the antibody are antibodies or antibody fragments. 7. The method according to claim 1-5, characterized in that the reactant with the antibody reactant protein _{A is} Staphylococcus aureus. 8. The method according to claim 1-5, characterized in that the reactant reacting with the antibody is a complement factor. 9. The method according to claim 8, characterized in that the reactant reacting with the antibody is an anti-complement antibody or an antibody fragment. 10. The method according to claim 1-6, characterized in that the reactant reacting with the antibody is an antibody against Fc fragment of immunoglobulin or against immunoglobulin aggregates or a rheumatoid factor. 11. The method according to claim 1, characterized in that the marking agent is a radioactive substance, an enzyme, a coenzyme or a fluorescent substance. 12. Diagnostic agent for antigen-specific immune complex determination, characterized in that it contains carrier-bound reactants specific for the antigen or the antibody of the immune complex.